Eukaryotic cells have developed innate mechanisms to protect them from invasion by viruses and foreign nucleic acids, most of which involve silencing of gene expression. One such adaptive innate response is RNA interference (RNAi). The RNAi pathway is highly conserved in almost all eukaryotes, and is involved in a variety of phenomena that negatively impact gene expression, including RNA silencing, microRNA processing, RNA-directed DNA methylation, histone H3K9 methylation, and the establishment of heterochromatin. In plants, RNAi is an important adaptive defense which is induced by and targets viruses. Plant viruses counter this attack by producing proteins that suppress components of the pathway, and these silencing suppressors were first identified as pathogenicity determinants. It is likely that mammalian viruses must also suppress RNAi, and their recently demonstrated susceptibility to RNAi-based approaches supports this view. Viral silencing suppressors represent potent targets for the development of antiviral and vaccine therapeutics, but the presence of such suppressors among mammalian viruses is unknown. Herpes simplex virus (HSV) is effective in establishing its transcription program in cells in which it replicates productively, but viral gene transcription is largely silenced during latency. The effectiveness with which HSV avoids being silenced in permissive cells makes HSV a strong candidate for encoding a silencing suppressor. It is hypothesized that HSV encodes one or more proteins that suppress the RNAi pathway in order to productively replicate. The ability of wild-type and mutant HSV to induce RNAi will be determined by the appearance of virus-specific small interfering RNA (siRNA) using a candidate screening approach and a global approach to clone small dsRNAs from infected cells. In aim 2, a transient silencing system will be used to identify silencing suppressors encoded by HSV.